Synapses are the essential components of neural circuits in the brain. It is widely accepted that a causal factor in many neuropsychiatric disorder is synaptic dysfunction, including abnormal synapse formation or defects in synaptic transmission. However, few tools are available for imaging synapse density and function in the living mammalian brain. Although viral delivery of genetically encoded probes (e.g. fluorescent proteins, channel rhodopsins) is widely used for live imaging/functional assays of synapses in the rodent brain, this approach is highly invasive, requires specialized techniques and reagents, and has had limited success in non-human primates, let alone humans. We therefore propose an alternative approach based upon the molecular recognition of native synaptic components by organic small molecules, thereby facilitating their selective localization to synapses. This chemical approach has the potential to extend the use of synaptic markers from rodent models into non-human primates and humans via non-invasive administration routes. Such synaptic markers could also serve as molecular targeting devices for the delivery of sensors and therapeutics to synapses in the living mammalian brain, affording transformative tools for neuroscience research, as well as for the diagnosis and treatment of neuropsychiatric disorders. As a first step toward these long-term goals, we propose in this application to develop a high-throughput screening (HTS) platform for discovery of small molecule fluorescent synaptic markers. We have obtained a library of ~8,000 novel fluorescent compounds based on diverse fluorophore structural cores, spanning a wide structural and spectroscopic range. This unique resource is a valuable tool for the discovery and development of synaptic markers. In Aim 1, we will develop a HTS assay using cultured cortical neurons in 96-well plate format. Synaptic labeling will be assessed based on colocalization with fluorescent protein (FP)-tagged synaptic vesicle-associated proteins that label presynaptic boutons. The screening, imaging, data mining, and hit selection protocols will be optimized with a preliminary screen of ~1,000-2,000 fluorescent compounds. In Aim 2, we will use protocols optimized in Aim 1 to screen the entire library of ~8,000 fluorescent dyes, and identify hit compounds that label synapses. We will subsequently determine their optimal concentrations and selectivity for glutamatergic versus GABAergic synapses. In Aim 3, we will perform a series of post-screening assays to eliminate toxic compounds and false positives. Select compounds will be resynthesized, structurally confirmed, and re-tested to validate their synaptic labeling. Remaining advanced hits will subsequently be classified as stable (fluorescence is stable during synaptic stimulation), ratiometric (light emission properties change during stimulation), or dynamic (compound is lost from synapses during exocytosis/synaptic activity) synaptic markers. Finally, their impact on synaptic function will be assessed using live imaging methods.